The present invention relates to a method for producing L-glutamic acid by fermentation accompanied by precipitation. L-Glutamic acid is widely used as a material for seasonings and so forth.
L-Glutamic acid is mainly produced by fermentative methods using so-called coryneform bacteria producing L-glutamic acid and belonging to the genus Brevibacterium, Corynebacterium or Microbacterium, or mutant strains thereof (Amino Acid Fermentation, pp. 195–215, Gakkai Shuppan Center, 1986). As methods for producing L-glutamic acid by fermentation by using other bacterial strains, there are known a method using a microorganism belonging to the genus Bacillus, Streptomyces, Penicillium or the like (U.S. Pat. No. 3,220,929), a method using a microorganism belonging to the genus Pseudomonas, Arthrobacter, Serratia, Candida or the like (U.S. Pat. No. 3,563,857), a method using a microorganism belonging to the genus Bacillus, Pseudomonas, Serratia, Aerobacter aerogenes (currently referred to as Enterobacter aerogenes) or the like (Japanese Patent Publication (Kokoku) No. 32-9393), a method using a mutant strain of Escherichia coli (Japanese Patent Application Laid-open (Kokai) No. 5-244970) and so forth. In addition, the inventors of the present invention have proposed a method for producing L-glutamic acid by using a microorganism belonging to the genus Klebsiella, Erwinia or Pantoea (Japanese Patent Application Laid-open No. 2000-106869).
Further, there have been disclosed various techniques for improving L-glutamic acid-producing ability by enhancing activities of L-glutamic acid biosynthetic enzymes through use of recombinant DNA techniques. For example, it has been reported that introduction of a gene coding for citrate synthase derived from Escherichia coli or Corynebacterium glutamicum was effective for enhancement of L-glutamic acid-producing ability in Corynebacterium or Brevibacterium bacteria (Japanese Patent Publication No. 7-121228). In addition, Japanese Patent Application Laid-open No. 61-268185 discloses a cell harboring recombinant DNA containing a glutamate dehydrogenase gene derived from Corynebacterium bacteria. Further, Japanese Patent Application Laid-open No. 63-214189 discloses a technique for improving L-glutamic acid-producing ability by amplifying a glutamate dehydrogenase gene, an isocitrate dehydrogenase gene, an aconitate hydratase gene and a citrate synthase gene.
Although L-glutamic acid productivity has been considerably increased by breeding of the aforementioned microorganisms or improvement of production methods, development of methods for more efficiently producing L-glutamic acid at a lower cost is required to respond to further increase of the demand in future.
There is known a method wherein fermentation is performed and crystallized L-amino acid is accumulated in the culture (Japanese Patent Application Laid-open No. 62-288). In this method, the L-amino acid concentration in the culture is maintained below a certain level by precipitating the accumulated L-amino acid in the culture. Specifically, L-tryptophan, L-tyrosine or L-leucine is precipitated during fermentation by adjusting the temperature and pH of the culture or by adding a surface active agent to the medium.
While a fermentative method involving a precipitating L-amino acid is known as described above, amino acids suitable for this method are those of relatively low water solubility, and no example of applying the method to highly water-soluble amino acids such as L-glutamic acid is known. In addition, the medium must have low pH to precipitate L-glutamic acid. However, L-glutamic acid-producing bacteria such as those mentioned above cannot grow under acidic conditions, and therefore L-glutamic acid fermentation is performed under neutral conditions (U.S. Pat. Nos. 3,220,929 and 3,032,474; Chao K. C. & Foster J. W., J. Bacteriol., 77, pp. 715–725 (1959)). Thus, production of L-glutamic acid by fermentation accompanied by precipitation is not known. Furthermore, it is known that growth of most acidophile bacteria is inhibited by organic acids such as acetic acid, lactic acid and succinic acid (Yasuro Oshima Ed., “Extreme Environment Microorganism Handbook”, p. 231, Science Forum; Borichewski R. M., J. Bacteriol., 93, pp. 597–599 (1967) etc.). Therefore, it is considered that many microorganisms are susceptible to L-glutamic acid, which is also anorganic acid, under acidic conditions, and there has been no report that search for microorganisms showing L-glutamic acid-producing ability under acidic conditions was attempted.